Peptide Tyrosinase Inhibitors and Uses Thereof

ABSTRACT

Disclosed are peptides, which inhibit the enzymatic activity of tyrosinase, as well as formulations and methods for their use in the reduction of skin pigmentation, and methods of administering the inhibitory peptides in a topical formulation. The peptides are characterized by sequences RADRADC and PLG-OH. Methods of skin treatment are also provided, the methods further including use of a peptide characterized by the amino acid sequence SFLLRN.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/937,331, filed on Jun. 27, 2007, which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

None.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

Applicants assert that the paper copy of the Sequence Listing isidentical to the Sequence Listing in computer readable form found on theaccompanying computer disk. Applicants incorporate the contents of thesequence listing by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of tyrosinase inhibitors andto methods and compositions of treatment involving inhibition of thisenzyme.

2. Related Art

The present invention relates to novel biological agents, specificallypeptides that reduce the enzymatic activity of tyrosinase. These agentshave use as research and development tools in basic scienceinvestigation, in diagnostic applications, as cosmeceuticals for thetreatment of skin conditions characterized by hyperpigmentation, and astherapeutics for the treatment of pathological conditions that rely ontyrosinase enzyme activity to promote their tumorigenicity.

Melanin plays an important role in protecting human body from theharmful effects of ultraviolet rays. Melanin is also an important factorin medical science and cosmetology. It is known that melanin is formedor synthesized in skin tissues. Excessive amounts of melanin darken theskin, and the nonuniform distribution of melanin causes chloasma andephelis, both of which are skin disorders. The biosynthesis pathway ofmelanin involves the catalytic hydroxylation of tyrosine toL-3,4-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA todopachrome. An effective way to inhibit the synthesis of melanin is toblock the hydroxylation of tyrosine.

Hydroquinone (HQ) has been used since the 1950s in commerciallyavailable over-the-counter skin lightener products and since the 1960sas a commercially available medical product. It is also used in cosmeticproducts such as hair dyes and products for coating finger nails.Beginning in 2001, HQ is no longer authorized for use in cosmetic skinlightening formulations in European Union countries, although productscontaining arbutin, an analogue of HQ, and botanicals, including plantsthat naturally contain HQ and arbutin, continue to remain available inEuropean countries See also, Matsubayashi et al., “Pharmaceutical andclinical assessment of hydroquinone ointment prepared by extemporaneousnonsterile compounding,” Biol Pharm Bull. 2002 January;25(1):92-6. Asdisclosed there, ointments of the skin depigmentation agent hydroquinone(HQ) have been prepared by extemporaneous nonsterile compounding inJapan by imitating skin lightening creams commercially available in theU.S.A. and European Union. However, various problems have been observedincluding chromatic aberration of HQ ointments, relatively largevariability of efficacy, and undesirable side effects although they weremild. HQ has a published IC50 of about 700 μM.

Therapies containing hydroquinone have been outlawed in Asian countries,making the standard HQ treatment inaccessible to a large number ofpeople suffering from this condition. In fact, the United States FDA hasissued a notice indicating that it may too ban the use of hydroquinonedomestically. Furthermore, hydroquinone has been associated withvisceral malignancy and long-term topical delivery may be a potentiallyharmful therapeutic option. Hydroquinone in the best of circumstancesleads to only a partial alleviation of hyperpigmentation. Somecosmeceutical formulations have included other active ingredients suchas kojic acid, arbutin, and vitamin C but efficacy has thus far beendisappointing due to problems with chemical instability or inability todeliver the active to the appropriate layer of skin. Although higherconcentrations have been utilized, patients often discontinue treatmentdue to skin irritation. This led to the addition of topical steroids inorder to reduce irritation from the active ingredients such as retin Aand hydroquinone. Since melasma and other hyperpigmentary disordersoften take months to years to treat, use of topical steroids on the faceat the strength required to combat irritant effects of activeingredients is not possible without causing topical steroid-induced sideeffects. When medium or greater potency topical steroids are used on theface for more than several weeks consecutively, skin atrophy, fragilityand telengiectasia commonly occur. This side effect profile isunacceptable, especially in areas such as the face.

Infrared lasers have been used with some success. They generally aremore effective for conditions that localize pigment to the deeper skinareas such as the dermis. In order to effectively treat the epidermis,an ablative treatment is usually employed. This therapy is associatedwith significant downtime for the patient, including creation ofsecond-degree bum or erosion leaving the patient susceptible toinfection. In addition, laser therapy is an expensive treatment optionthat many patients cannot afford. In extreme cases, depigmentation ofthe skin has been elected when bleaching agents have been unsuccessful.Numerous pathological conditions can lead to the deposition of pigmentinto the skin aberrantly. For example, it is well known that hormonalimbalance can cause facial and extremity hyperpigmentation, mostfrequently observed in women during or following pregnancy. Often times,this hyperpigmentation becomes aesthetically disfiguring, leading toproblems with self-esteem and embarrassment in social situations.Melasma often times affects individuals with Fitzpatrick type IV-VIskin. This constitutes a significant portion of the worldwidepopulation.

A large number of individuals with Fitzpatrick type IV to VI skin are ofAsian descent.

According to the Fitzpatrick skin type scale, based on a test ofappearance and skin reaction to sun exposure, individuals are generallycategorized as follows:

Type I: Very fair skin tone, blond or redhead,

Type II: Light skin tone, will tan, but usually burns.

Type III: White to olive skin tone, sometimes burns.

Type IV: Medium brown skin tone, rarely bums.

Type V: Dark brown skin tone, very rarely burns.

Type VI: Black skin tone, very dark eyes, burn resistant.

In addition to melasma, hyperpigmentation of aesthetically sensitivelocations such as the face may take place after inflammation due todisorders such as acne or rosacea, amongst others. These conditions mayalso lead to significant psychological discomfort. In the United States,$13 billion are spent on cosmeceuticals each year. With the anticipatedban of hydroquinone in the US market, in conjunction with the continuedstability (vitamin C) or delivery (arbutin, kojic acid, etc) problems ofother non-pharmacological agents currently on the market, oligopeptideinhibitors may provide a solution to this large unmet need.

Specific Patents and Publications

Scot et al., “Production of cyclic peptides and proteins in vivo,” Proc.Nat. Acad. Sci. Vol. 96, Issue 24, 13638-13643, Nov. 23, 1999, disclosesthe production of the cyclic, eight-amino acid tyrosinase inhibitorpseudostellarin F in bacteria.

Verma et al., “Modulation of agonist binding to human dopamine receptorsubtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog,”J Pharmacol Exp Ther. 2005 December;315(3):1228-36. Epub 2005 Aug. 26,discloses the role of the hypothalamic tripeptideL-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrainedanalog3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide(PAOPA) in modulating agonist binding to human dopamine (DA) receptorsubtypes.

U.S. Pat. No. 6,165,982 to Yamada, et al., issued Dec. 26, 2000 entitled“Use of sericin as antioxidants and tyrosinase inhibitors,” discloses acomposition useful as an antioxidant or an inhibitor for tyrosinaseactivity which comprises as an active ingredient a sufficient amount ofsericin to exert an antioxidizing ability. Sericin is a high molecularweight, natural, soluble glycoprotein constituent of silk. Sericin bindsto the keratin of skin and hair, forming a protective film.

U.S. Pat. No. 5,126,327 to Takeuchi, et al., issued Jun. 30, 1992,entitled “Melanocyte-stimulating hormone inhibitor and externalpreparation containing the same,” discloses a melanocyte-stimulatinghormone inhibitor which has certain amino acid sequences, an acyl grouphaving 1 to 12 carbon atoms, an amino acid residue, or acylatedderivative thereof having 1 to 12 carbon atoms, peptide residue having 2to 40 amino acid residues or acylated derivative thereof.

U.S. Pat. No. 7,025,957 to Arquette, issued Apr. 11, 2006, entitled“Composition and method to whiten skin,” discloses a compositioneffective as a skin whitening agent. The composition includesSimmondsin, which is a glycoside extracted from jojoba meal (Simmondsiachinensis). In certain embodiments, the composition comprises an extractof jojoba (Simmondsia chinensis). The composition is administered bytopically applying to an individual a formulation in an amount effectiveto whiten skin, where that composition comprises a jojoba extract.

U.S. Pat. No. 7,083,781 to Fotinos, et al., issued Aug. 1, 2006,entitled “Film forming polymers, methods of use, and devices andapplications thereof,” discloses compositions and methods for deliveringactive agents to the skin of a subject, including a polymer, an activeingredient and a solvent, the compositions being capable of delivery byrolling, spreading, aerosol or in droplets and of forming a film incontact with the skin. A cosmetic active agent known in the art may beincorporated in the film forming compositions for improving skinappearance. Anti-hyperpigmentation agents typically used forcounterbalancing this condition can include tyrosinase inhibitors suchas peptide mixtures and plant extracts, fermentation products, andantioxidants such as hydroquinone, kojic acid, ascorbic acidderivatives, synthetic or natural derivatives of hydroquinone andhydroquinone precursors. In preferred embodiments of the invention,anti-hyper pigmentation agents are Melawhite of Pentharm Ltd., Basel,Switzerland; Biowhite™ of Coletica, France; Etioline of Sederma, France;Arbossa of Kelesima, Italy; Gatuline whitening of Gattefosse, France;Ascorbocilan C of Exsymol, Monaco; and Kojic acid of Alps Pharm., Japan.

U.S. Pat. No. 7,125,572 to Lee, issued Oct. 24, 2006, entitled“Tyrosinase inhibitor extract,” discloses a tyrosinase inhibitor extractfrom lemon peels. The tyrosinase inhibitor provides advantageous skinwhitening effects. According to the invention, the tyrosinase inhibitorextract of the invention has a main absorbance at 280 nm. This indicatesthat the tyrosinase inhibitor extract contains a protein or peptide. Itis believed that the protein or peptide is the main active component forinhibiting tyrosinase. The other components of the extract may provideadditional effects such as anti-aging and anti-oxidation. The tyrosinaseinhibitor extract can be prepared to be in various forms, includinglotions, emulsions, creams, ointments, sticks, solutions, packs, andgel. The tyrosinase inhibitor extract may be admixed with anyingredients ordinarily used in cosmetics, such as oily substances,humectants, thickeners, preservatives, emulsifiers, medical ingredients,perfumes, emulsification stabilizers and the like.

Various other patents and publications disclose peptides of unrelateduses. See, e.g., U.S. Pat. No. 6,982,249, disclosing TRAP: thrombinreceptor activation peptide (in connection with SEQ ID NO:1).

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present invention concerns certain peptide sequences exemplified as:

>1 SEQ ID NO: 1 SFLLRN (“SF peptide”) >2 SEQ ID NO: 2 RADSRADC (“RApeptide”) >SEQ ID NO: 3 (comparative example) (“VL peptide”) VLLK

In addition, the tripeptide PLG-OH (“PLG”) was used, and a control,poly-L lactic acid.

In addition, variations to the exact sequence RADSRADC may be made, suchas

>SEQ ID NO: 4: RGDSRGDC.

The present invention is further directed to kits and compositionscontaining the present peptides, and methods of treatment of conditionsinvolving expression of tyrosinase, in which the present peptides areadministered topically for the treatment of conditions involvingmelanocyte activity in the skin and may be administered internally. Itis noted that the PLG peptide contains native amino and carboxy termini.It is denoted as PLG-OH, indicating a native carboxy terminus. This isdistinguished from the known peptide PLG-NH₂, having a glycine amideterminus.

Furthermore, the present peptides are less irritable to human skin andcan replace hydroquinone (HQ) in topical applications, resulting in aformulation, which is substantially free of HQ.

The present invention is further directed to kits and compositionscontaining the present peptides, and methods of treatment of conditionsinvolving expression of tyrosinase, in which the present peptides areadministered topically for the treatment of conditions involvingmelanocyte activity in the skin. Other formulations are useful intreating tyrosinase activity in other regions of the body and may beadministered internally.

Thus, in certain aspects, the present invention is directed to apurified peptide having at least 63% identity to an amino acid sequenceRADSRADC and having an IC 50 of tyrosinase of less than about 10 mM. Thepeptide in effect may be substituted in up to three positions, includingdeletions. Guidance is given for making these alterations, as well astesting them for inhibitory potency. In some cases, the peptide willhave an IC 50 of less than about 5 mM, or even less. In some cases, itis within the present invention to substitute a residue with R or F, inthat these residues serve to increase peptide binding to the tyrosinase.Between one and three residues may be substituted with V, A, L, M or I,in that these residues serve to increase inhibitory function.

In addition, one may substitute K with L or R; F with W or Y; E with D,as it is known that these residues are similar. It is also advantageousin some embodiments to have two adjacent charged amino acids. In certainaspects, the invention comprises the use of D-amino acids for some orall of the amino acids. The peptides may be linked to a modulatinggroup, as defined below, such as a palmitic acid or ester.

In one aspect, the substitutions may be presented as a chart:

Column 1-recited residue Column 2-substitute R K or L S T, A, A, L, M, ID N, V, A, L, M, I C V, A, L, M, I A G, V, A, L, M, I L I, V, A, M, I ND F R, W, Y

In certain aspects, the present invention comprises a topicalformulation useful in skin whitening. The formulation is made fromdermatologically acceptable ingredients. The formulation may comprisestandard carrier material, as well as in certain cases, a secondarytreatment agent and a peptide substantially identical to a sequenceselected from the group consisting of PLG-OH, RA peptide and YR peptide.These peptides may be altered as described above. In certainembodiments, the formulations may be adapted for potential over thecounter use, or for prescription use. For over the counter use, one mayemploy a formulation wherein the peptide is at a concentration less thanabout two times the IC 50 concentration. For pharmaceutical use, one maythe peptide may be at a concentration of two to 100 times the IC 50concentration.

The present peptides are in certain aspects superior to HQ, and may beformulated to be substantially free of HQ. In certain formulations,different peptides may be combined, with different sequences, differentattached groups, and so forth. The carrier may include a materialselected from: hydrating formulations, antioxidant formulations, andfree radical scavengers.

In certain formulations, the peptides will have improved skin uptake bybeing formulated in liposomes.

In certain aspects, the present invention comprises methods of skintreatment, involving skin lightening (whitening). This aspect includes amethod for treatment of skin comprising administering to the skin apeptide substantially identical to one of: RADSRADC, SFLLRN or thepeptide PLG-OH, wherein said administering of the peptide inhibitstyrosinase sufficiently to lighten skin pigmentation. The administeringmay comprise administering a topical preparation, as referred to above,and may further comprise a secondary treatment product.

The present methods of treatment also include carrying out the skinwhitening with further assistance from a microdermabrasion process. Theadministering may be simultaneous with the microdermabrasion process.The administering is in conjunction with a radiation process. Suchprocesses may be used to increase skin permeability. Furthermore, theadministering may be in conjunction with a physical treatment carriedout by an abrading device a microneedle, an electroporation device, oran iontophoretic device.

In certain aspects, the present invention comprises a kit for carryingout a skin whitening procedure, comprising a purified peptide having anIC 50 of tyrosinase of less than about 10 mM and selected from the groupconsisting of: a peptide having and a sequence at least 63% identical tothe sequence of either RA or SF peptide; a dermatologically acceptablecarrier; a secondary treatment product; and directions for use. The kitmay be aimed at consumers or physicians, and may include aprecombination of the peptide and carrier, such that the formulation isready to apply, or it may require mixing of the peptide with thecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing in vitro effects of peptides of RA peptide,SEQ ID NO: 2, and PLG-OH on tyrosinase activity.

FIG. 2 is a graph showing in vitro effects of peptide of SF peptide, SEQID NO: 1, VL Peptide, and poly-L lactic acid control on tyrosinaseactivity.

FIG. 3 is a bar graph showing percent improvement in facial pigmentationof vehicle vs. PLG-OH.

FIG. 4 is a bar graph showing proliferation rate of cells exposed tocontrol, tyrosinase inhibitors HQ and RA peptide, designated on thegraph as “P3.”

FIG. 5 is a bar graph showing inhibition of melanin production in cellsunder different concentrations of inhibitors HQ and RA peptide (“P3”).

FIG. 6 is a bar graph showing tyrosinase activity of differentconcentrations of inhibitors HQ and RA peptide (“P3”).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview

Short peptides of between about 4 to 10 amino acids, are disclosed andshown to have inhibitory activity against tyrosinase. Short sequencepeptides are synthetically designed using naturally occurring aminoacids, and therefore are biologically safe. They can be delivered tomelanocytes through a number of mechanisms, including but not limited toliposomes, allowing access to the appropriate skin layer. These peptidesdo not suffer from oxidation problems, as does the most commonly usedingredient vitamin C. These peptides do not cause liver cancer, as doeshydroquinone, and since they are derived from naturally occurring aminoacids, are easily degraded intracellularly upon inactivation oftyrosinase. They cause skin lightening or whitening by inhibitingmelanin synthesis.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The term “tyrosinase” is used herein to refer to monophenolmonooxygenase (EC 1.14.18.1; CAS number: 9002-10-2), an enzyme thatcatalyses the oxidation of phenols (such as tyrosine). It is acopper-containing enzyme present in plant and animal tissues thatcatalyzes the production of melanin and other pigments from tyrosine byoxidation. All tyrosinases have in common a binuclear type 3 coppercenter within their active site. Here two copper atoms are eachcoordinated with three histidine residues. Matoba et al.,“Crystallographic evidence that the dinuclear copper center oftyrosinase is flexible during catalysis,” J Biol Chem. 2006 Mar.31;281(13):8981-90. Epub 2006 Jan. 25, disclose a three-dimensionalmodel of a tyrosinase catalytic center.

The term “peptide” is used herein in its conventional sense, i.e., apolymer in which the monomers are amino acids and are joined togetherthrough amide bonds, alternatively referred to as a polypeptide. Whenthe amino acids are α-amino acids, either the L-optical isomer or theD-optical isomer may be used. Additionally, unnatural amino acids, forexample, β-alanine, phenylglycine and homoarginine are also meant to beincluded. The present peptides are two or more amino acid monomers longand may be up to 20 amino acid monomers long. Standard abbreviations foramino acids are used (as described below).

The term “carrier” refers to compounds commonly used on the formulationof pharmaceutical compounds used to enhance stability, sterility anddeliverability of the therapeutic tyrosinase inhibitor. When the peptidedelivery system is formulated as a solution or suspension, the deliverysystem is in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

The term “topical” or “topically” is used here in its conventional senseas referring to a spot, which can be in or on any part of the body,including but not limited to the epidermis, any other dermis, or anyother body tissue. Topical administration or application means thedirect contact of the peptide with tissue, such as skin or membrane,which contains melanin-producing cells. Methods of applying the presenttopical agents to the skin or mucosa include “non-finite” or liquid orsemi-liquid carriers such as gels, lotions, emulsions, creams, plasters,or ointments, or “finite” carriers, non-spreading substances whichretain their form, e.g., patches, dressings and bandages. The solventsfor the finite and non-finite forms of the active peptides arenon-toxic, pharmaceutically acceptable substances, preferably liquids,which do not substantially negatively affect the adhesion properties orsolubility of the system. The solvent is preferably a polyhydric alcoholor combination of polyhydric alcohols. The term polyhydric alcohol meansany organic polyalcohol and includes dipropylene glycol, propyleneglycol, polyethylene glycol, glycerin, butylene glycol, hexylene glycol,polyoxyethylene, polypropylene glycol, sorbitol, ethylene glycol, andthe like. Other suitable solvents include fatty acids such as oleicacid, linoleic acid, capric acid and the like, as well as fatty estersor alcohols. Further suitable solvents include other non-toxic,non-volatile solvents commonly used in dermal or transdermalcompositions for dissolving peptide-based compositions.

The term “sequence identity” in the context of two polypeptide sequencesrefers to the residues in the two sequences, which are the same whenaligned for maximum correspondence. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homologyalignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson and Lipman Proc. Natl.Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection. Sequence identity may be calculated onthe basis of residues identical to a reference sequence. For example,for RADSRADC, having 8 residues, one may have 5 identical residues andhave 5/8 or 63% sequence identity. Because of the limited length of thepeptides, at least 63% identity is considered “essentially identical”when changes are made according to the present teachings. One may alsohave 6/8 (75%) or 7/8 (88%) sequence identity. As a further example, aresidue may be eliminated, such as the cysteine, or an R may be changedto V, L, M or I and one would have and have 7/8 or 88% identity.

The terms “substantial identity” as used herein denotes a characteristicof a polypeptide sequence, wherein the polypeptide comprises a sequencethat has at least 60 percent sequence identity, preferably at least 85percent identity and often 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of the entire peptide length.Substantial identity further involves a conservative substitution of anamino acid. The term “essentially identical” in the context of thepresent 6 or 8 residue peptides means that two of six or three of eightamino acid substitutions are permitted, according to the presentteachings specifically providing guidance in making substitutions, andthe definition above.

As further guidance in making amino acid substitutions, one maysubstitute by changing a given residue to R, or F to increase bindingproperties, or change it to V, A, L, M or I to increase inhibitoryproperties. One may direct one part of the peptide to binding to thetyrosinase enzyme, and another part towards inhibition. It is generallypreferred not to change F or R and to change K or E, as well as Y or W.Changes to Y should be considered in the context of the overallsequence, since t is the residue that is the natural substrate fortyrosinase. It should be noted that some changes may in fact result inan increase of tyrosinase activity. See, for further guidance, Schurinket al., “Novel peptides with tyrosinase inhibitory activity,” Peptides28:485:495 (January 2007).

Conservative amino acid substitutions are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids are generally divided into families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. More preferredfamilies are: serine and threonine are aliphatic-hydroxy family;asparagine and glutamine are an amide-containing family; alanine,valine, leucine and isoleucine are an aliphatic family; phenylalanine,tryptophan, and tyrosine are an aromatic family, and cysteine andmethionine as a sulfur-containing side chain family. For example, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Preferredconservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic acid-aspartic acid, cysteine-methionine, andasparagine-glutamine.

The term “keratinous tissue,” as used herein, refers tokeratin-containing layers disposed as the outermost protective coveringof mammals (e.g., humans, dogs, cats, etc.) which includes, but is notlimited to, skin, mucosa, lips, hair, toenails, fingernails, cuticles,hooves, etc.

The term “topical application,” as used herein, means to apply or spreadthe compositions of the present invention onto the surface of thekeratinous tissue.

The term “dermatologically-acceptable,” as used herein, means that thecompositions or components thereof so described are suitable for use incontact with mammalian keratinous tissue without undue toxicity,incompatibility, instability, allergic response, and the like.

The term “injectable formulation,” as used herein, means a formulationsuitable for injection into humans and/or animals, wherein the injectionis intradermal, subcutaneous, intramuscular or intravenous. Theseformulations will be sterile, pyrogen free, and at a physiologicallyacceptable pH.

The term “radiation process,” as used herein, means a treatment processas applied to a subject's skin or internal tissue, and is used forcosmetic or therapeutic purposes. The term includes the use ofelectromagnetic radiation devices, such as lasers, LEDs, radiofrequency,etc. The term also includes the use of ultrasound devices. These devicesall are used in processes in which skin whitening using the presentagents may be carried out. Some of these processes alter the stratumcorneum permeability, and would be beneficially useful in a process ofadministering the present peptides.

The term “IC 50,” as is understood in the art, means the means theconcentration of tyrosinase inhibitor peptide required to effect 50%inhibition of tyrosinase activity, as conducted in an in vitro assay; avalue of “less than” a certain concentration includes IC 50 values atlower concentrations. The term about may encompass plus or minus 10%variation, and variations resulting from different reagents,experimental conditions, etc. In vitro determinations of IC 50 using apurified tyrosinase preparation (e.g., mushroom tyrosinase) are usefulin determining a clinical dose.

General Method and Materials

The present materials and methods in a general sense are peptides, whichinhibit tyrosinase activity and may be formulated for application tohumans. They thus are useful in treatment or amelioration of conditionsinvolving over production of melanin.

Peptides

The present peptides include peptide analogues or peptide derivatives orpeptidomimetics that retain the ability to inhibit a tyrosinase activitywithin a cell. For example, an inhibitory peptide tyrosinase modulatorof the invention may be modified to increase its stability,bioavailability, solubility, etc. The terms “peptide analogue,” “peptidederivative,” and “peptidomimetic” are used herein to include moleculesthat mimic the chemical structure of a peptide and retain the functionalproperties of the peptide. Approaches to designing peptide analogs areknown in the art. For example, see Farmer, P. S. in Drug Design (E. J.Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp.119-143; Ball.J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A.and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues,derivatives and peptidomimetics include peptides substituted with one ormore benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942), peptides with methylated amide linkages and“retro-inverso” peptides (see U.S. Pat. No. 4,522,752 by Sisto). Peptideanalogues, peptide derivatives and peptidomimetic are described infurther detail below.

Peptides of the present invention may comprise residues from any of thenaturally occurring amino acids, or from non-naturally-occurring aminoacids. These naturally occurring and non-naturally-occurring amino acidsmay be in the D or L configuration. The terms D and L are used herein asthey are known to be used in the art. Peptides of the invention includesingle amino acids and short spans (e.g., 1-10) of amino acids. Inaddition, modified peptides of the present invention may also comprise amonomer or dimer.

The standard single letter and three letter codes for amino acids areused herein and are as follows:

A (Ala) Alanine C (Cys) Cysteine D (Asp) Aspartic acid E (Glu) Glutamicacid F (Phe) Phenylalanine G (Gly) Glycine H (His) Histidine I (Ile)Isoleucine K (Lys) Lysine L (Leu) Leucine M (Met) Methionine N (Asn)Asparagine P (Pro) Proline Q (Gln) Glutamine R (Arg) Arginine S (Ser)Serine T (Thr) Threonine V (Val) Valine W (Trp) Tryptophan Y (Tyr)Tyrosine

As described above, the indicated residues may be the naturallyoccurring L amino acid, or a modification thereof, that is, a chemicalmodification, an optical isomer, or a link to a modifying group. It iscontemplated that specific modifications may be made within the peptidethat maintain the ability of the present peptides to specificallyinhibit the activity of tyrosinase whereby it catalyzes the first twosteps in the pathway for pigment synthesis: hydroxylation of the aminoacid tyrosine into dihydroxyphenylalanine (DOPA) and/or the subsequentoxidation into dopaquinone

It is also contemplated that specific modifications may be made in aparticular sequence in order to confer some additional desirableproperty to the peptide. Certain amino acids may be substituted forother amino acids in a protein structure without appreciable loss ofpeptide activity. Since it is the interactive capacity and nature of apeptide that defines that peptide's biological functional activity,certain amino acid sequence substitutions can be made even in a shortpeptide sequence and nevertheless obtain a peptide with like properties.It is thus contemplated by the inventor that various changes may be madein the sequence of the present tyrosinase inhibitors without appreciableloss of biological utility or activity and perhaps may enhance desiredactivities.

For example, in designing peptide constructs with tyrosinase inhibitoryproperties, substitutions may be used which modulate one or moreproperties of the molecule. Such variants typically contain the exchangeof one amino acid for another at one or more sites within the peptide.For example, certain amino acids may be substituted for other aminoacids in a peptide structure in order to enhance the interactive bindingcapacity of the structures. One may also substitute D- for L-aminoacids, or include certain side chain covalent modifications.

In making such changes, the hydropathic index of amino acids may beconsidered.

The importance of the hydropathic amino acid index in conferringinteractive biologic function on a protein is generally understood inthe art (Kyte and Doolittle, 1982). It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

In modifying the presently exemplified sequences, certain amino acidsmay be substituted by other amino acids having a similar hydropathicindex or score and still result in a protein with similar biologicalactivity, i.e., still obtain a biological functionally equivalentprotein. In making such changes, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those that are within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

Substitution of like amino acids can also be made effectively on thebasis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein byreference, states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In modifying the exemplified sequences, amino acid substitutions mayalso be generally based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like but may nevertheless be madeto highlight a particular property of the peptide. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine, which, with histidine, are basic at physiologicalpH; glutamate and aspartate (which are acidic); serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

The naturally occurring amino acid side chains are illustrated below, inwhich * represents the attachment point to the compound's backbone:

The amino acids of the peptides of the present invention may also bemodified so that amino groups may be acylated, alkylated or arylated.Benzyl groups may be halogenated, nitrosylated, alkylated, sulfonated oracylated.

Various chemically modified amino acids may be incorporated into thepresent peptides. Examples of these include:

Acetylated

N-acetyl-L-alanine, N-acetyl-L-arginine; N-acetyl-L-asparagine;N-acetyl-L-aspartic acid; N-acetyl-L-cysteine; N-acetyl-L-glutamine;N-acetyl-L-glutamic acid; N-acetylglycine; N-acetyl-L-histidine;N-acetyl-L-isoleucine; N-acetyl-L-leucine; N2-acetyl-L-lysine;N6-acetyl-L-lysine; N-acetyl-L-methionine; N-acetyl-L-phenylalanine;N-acetyl-L-proline; N-acetyl-L-serine; N-acetyl-L-threonine;N-acetyl-L-tryptophan; N-acetyl-L-tyrosine; N-acetyl-L-valine.

Amidated

L-alanine amide, L-arginine amide

Formylated N-formyl-L-methionine Hydroxylated

4-hydroxy-L-proline

Lipid Modified S-farnesyl-L-cysteine, S-geranylgeranyl-L-cysteine,N-palmitoyl-L-cysteine,

S-palmitoyl-L-cysteine, N-myristoyl-glycine, N6-myristoyl-L-lysine

Methylated

N-methyl-L-alanine, N,N,N-trimethyl-L-alanine,omega-N,omega-N-dimethyl-L-arginineL-beta-methylthioaspartic acid, N5-methyl-L-glutamine, L-glutamic acid5-methyl ester3′-methyl-L-histidine, N6-methyl-L-lysine, N6,N6-dimethyl-L-lysine,N6,N6,N6-trimethyl-L-lysine, N-methyl-L-methionine,N-methyl-L-phenylalanine

Phosphorylated

omega-N-phospho-L-arginine, L-aspartic 4-phosphoric anhydride,S-phospho-L-cysteine,1′-phospho-L-histidine, 3′-phospho-L-histidine, O-phospho-L-serine,O-phospho-L-threonine, O4′-phospho-L-tyrosine

Other

L-selenocysteine, L-selenomethionine, L-3-oxoalanine,2-pyrrolidone-5-carboxylic acid, L-glutamyl5-glycerylphosphorylethanolamine,2′-[3-carboxamido-3-trimethylammonio)propyl]-L-histidine (diphthamide),N6-biotinyl-L-lysine, N6-(4-amino-2-hydroxybutyl)-L-lysine (hypusine),N6-retinal-L-lysine

Other modifications to the amino acids contained in the present peptidesare known in the art, and described, for example in Kuhner et al. U.S.Pat. No. 6,858,581, which describes chemically modified antimicrobialpeptides.

Modulating Groups

In a tyrosinase modulator of the invention having the formula shownabove, a modulating group for improved cellular uptake or efficacy orformulation may be attached directly or indirectly to the peptide of thetyrosinase inhibitor. For example, the modulating group can be directlyattached by covalent coupling to the peptide or the modulating group canbe attached indirectly by a stable non-covalent association. In oneembodiment of the invention, the modulating group is attached to theamino-terminus of the peptide of the modulator. Alternatively, inanother embodiment of the invention, the modulating group is attached tothe carboxy-terminus of the peptide of the modulator.

In yet another embodiment, the modulating group is attached to the sidechain of at least one amino acid residue of the peptide of the compound(e.g., through the epsilon amino group of a lysyl residue(s), throughthe carboxyl group of an aspartic acid residue(s) or a glutamic acidresidue(s), through a hydroxy group of a tyrosyl residue(s), a serineresidue(s) or a threonine residue(s) or other suitable reactive group onan amino acid side chain). Further guidance on preparing such modulatinggroups is found in U.S. Pat. No. 5,854,204.

The present peptides may also be conjugated to other tyrosinaseinhibitors such as kojic acid (C6H6O4;5-hydroxy-2-(hydroxymethyl)-4-pyrone) or gnetol (see Biosci BiotechnolBiochem. 2003 March;67(3):663-5.)

Another modulating group for enhancing cell permeability is an aminoacid sequence, which is recognized and taken up by melanocytes. D'Ursiet al., “A Membrane-Permeable Peptide Containing the Last 21 Residues ofthe GS Carboxyl Terminus Inhibits GS-Coupled Receptor Signaling inIntact Cells: Correlations between Peptide Structure and BiologicalActivity,” Mol Pharmacol 69:727-736, 2006 disclose cell-penetratingpeptides which are able to transport covalently attached cargoes such aspeptide or polypeptide fragments of endogenous proteins across cellmembranes. The authors coupled their peptide to the 16-residue fragmentpenetratin, and such fragment may be coupled to the peptides disclosedhere.

Thus, the term modulating group means a small organic molecule linked tothe peptide to affect its activity, either by improving its stabilityuptake or the like, or by providing additional tyrosinase inhibition.

In a preferred embodiment, the modifying group(s) comprises a cyclic,heterocyclic or polycyclic group. The term “cyclic group,” as usedherein, is intended to include cyclic saturated or unsaturated (i.e.,aromatic) group having from about 3 to 10, preferably about 4 to 8, andmore preferably about 5 to 7, carbon atoms. Exemplary cyclic groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcyclooctyl. Cyclic groups may be unsubstituted or substituted at one ormore ring positions. Thus, a cyclic group may be substituted with, e.g.,halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles,hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,sulfonates, selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or thelike.

In another preferred embodiment, the modulating group comprises a fattyacid bonded to the peptide, in order to increase uptake through theskin. Suitable fatty acids (which are meant to include the correspondingester) include fatty acid ester emollient selected from the groupconsisting of methyl palmitate, methyl stearate, isopropyl laurate,isopropyl myristate, isopropyl palmitate, ethylhexyl palmitate, lauryllactate and cetyl lactate.

The term “heterocyclic group” is intended to include cyclic saturated orunsaturated (i.e., aromatic) group having from about 3 to 10, preferablyabout 4 to 8, and more preferably about 5 to 7, carbon atoms, whereinthe ring structure includes about one to four heteroatoms. Heterocyclicgroups include pyrrolidine, oxolane, thiolane, imidazole, oxazole,piperidine, piperazine, morpholine. The heterocyclic ring can besubstituted at one or more positions with such substituents as, forexample, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, otherheterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides,phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, —CF₃,—CN, or the like. Heterocycles may also be bridged or fused to othercyclic groups.

Formulations

The peptides of the present invention are preferably formulated intotopical compositions that contain a dermatologically acceptable carrier.The phrase “dermatologically-acceptable carrier”, as used herein, meansthat the carrier is suitable for topical application to the keratinoustissue, has good aesthetic properties, is compatible with the actives ofthe present invention and any other components, and will not cause anyuntoward safety or toxicity concerns. A safe and effective amount ofcarrier is from about 50% to about 99.99%, preferably from about 80% toabout 99.9%, more preferably from about 90% to about 98%, and even morepreferably from about 90% to about 95% of the composition.

The carrier can be in a wide variety of forms. For example, emulsioncarriers, including, but not limited to, oil-in-water, water-in-oil,water-in-oil-in-water, and oil-in-water-in-silicone emulsions, areuseful herein.

Preferred carriers contain an emulsion such as oil-in-water emulsions,water-in-oil emulsions, and water-in-silicone emulsions.

Emulsions according to the present invention generally contain asolution as described above and a lipid or oil. Lipids and oils may bederived from animals, plants, or petroleum and may be natural orsynthetic (i.e., man-made). Preferred emulsions also contain ahumectant, such as glycerin. Emulsions will preferably further containfrom about 0.01% to about 10%, more preferably from about 0.1% to about5%, of an emulsifier, based on the weight of the carrier. Emulsifiersmay be nonionic, anionic or cationic. Suitable emulsifiers are disclosedin, for example, U.S. Pat. No. 3,755,560, issued Aug. 28, 1973, Dickertet al.; U.S. Pat. No. 4,421,769, issued Dec. 20, 1983, Dixon et al.; andMcCutcheon's Detergents and Emulsifiers, North American Edition, pages317-324 (1986).

The emulsion may also contain an anti-foaming agent to minimize foamingupon application to the keratinous tissue. Anti-foaming agents includehigh molecular weight silicones and other materials well known in theart for such use.

Suitable emulsions may have a wide range of viscosities, depending onthe desired product form. Exemplary low viscosity emulsions, which arepreferred, have a viscosity of about 50 centistokes or less, morepreferably about 10 centistokes or less, still more preferably about 5centistokes or less.

Preferred water-in-silicone and oil-in-water emulsions are described ingreater detail in US PGPUB 20060188462 by Bissett et al., published Aug.24, 2006, entitled “Skin care compositions containing a sugar amine.”

The present peptides may be formulated in liposomes. The presentpeptides may be contained in liposomes according to methods, forexample, as described in U.S. Pat. No. 4,944,948 to Uster, et al.,entitled “EGF/Liposome gel composition and method,” where onesubstitutes inhibitory peptide for the EGF used there. As describedthere, a high-viscosity aqueous dispersion of negatively chargedliposomes may be prepared with liposome-entrapped peptide. Thepeptide/liposome composition is formed by suspending a lipid mixturetypically containing equimolar amounts of neutral and negatively chargedphopholipids and cholesterol in a low-conductivity aqueous mediumcontaining peptide and a zwitterionic compound whose isoelectric pointis between pH 5.5 and 8.5 to form a gel-like composition. Furtherexemplary guidance may be found in U.S. Pat. No. 4,485,054 to Mezei, etal., entitled “Method of encapsulating biologically active materials inmultilamellar lipid vesicles (MLV).”

The present peptide tyrosinase inhibitors may also be prepared as anoral or injectable formulation. The pH of the injectable formulation isimportant, especially in regard to safety and comfort during injection,and especially if the preparation is supplied in a liquid formulation. Asuitable formulation may contain preservatives, such as sodium benzoate,methylparaben and propylparaben, etc., and may have a pH of 6.8-8.0 at25° C. The pH is preferably maintained by a buffer. Suitable bufferingagents include acetate buffers, 2-amino-2-methyl-1-propanol, glycinebuffers, phosphate buffers, (tris>hydroxymethyl-aminomethane) (TRIS)buffers, (2->N-morpholino-ethanesulfonic acid), etc. The formulationwill typically also comprise a carrier as defined above. Injectableformulations are suitable for use in the treatment of melanomas andother cancers that derive from cells expressing tyrosinase, e.g.,glioblastomas. Further details may be found in U.S. Pat. No. 5,773,291to Bouchard, et al., issued Jun. 30, 1998, entitled “Non-melanotyticmammalian cell constitutively expressing biologically active humantyrosinase and use thereof.” These formulations are useful withmelanocytes not approachable by topical application, such as melanocytesfound in non-keratinous tissue. Melanocytes are found in the basal layerof the epidermis as well as in hair follicles, the retina, uveal tract,and leptomeninges. These cells are the sites of origin of melanoma.Regarding oral formulations, an exemplary formulation may be found in US2007/0134279.

The present peptide tyrosinase inhibitors may be used alone or incombination with each other. They may also be used in combination withother biologically active drugs or cosmeceuticals. They may be deliveredby liposomes or other transdermal delivery mechanism, such as disruptivedevices etc. A fatty acid chain may be conjugated to the C-terminus orN-terminus of the peptide to promote non-liposomal based delivery vialipid partition into the stratum corneum. Other lethal or suicide agentsmay be conjugated to the peptides allowing delivery of a lethal orsuicide agent to those cells that express tyrosinase at high levels,such as melanoma cells.

Lipid peptide formulations of the present peptides are further describedin U.S. Pat. No. 6,287,590 to Dasseux, issued Sep. 11, 2001, entitled“Peptide/lipid complex formation by co-lyophilization;” U.S. Pat. No.5,543,389 to Yatvin, et al., issued Aug. 6, 1996, entitled “Covalentpolar lipid-peptide conjugates for use in salves,” and other references.

The length of the oligopeptides described herein, i.e., 20, orpreferably 12 or less amino acids, with biological inhibitory activityagainst tyrosinase, has not been previously described. To date, it isbelieved that all agents used to inhibit tyrosinase enzyme activity vivoare non-peptide based.

Further guidance on formulating the present peptides may be found in US20040086560 by Chaudhuri, et al., published May 6, 2004, entitled“Skin-lightening.”

The present peptides may further be formulated with other ingredientsuseful in treating or ameliorating skin conditions, or with ingredientsthat reduce irritation when the peptides are administered in conjunctionwith an abrasive procedure. Examples of these additional ingredients,termed here “secondary treatment agents,” include 1 percent vitamin K,and 1 percent hydrocortisone in a aqueous base); acne treatmentformulations (e.g., salicylic acid, alcohol base buffered by witchhazel, etc.); fine lines/wrinkle treatment formulations (e.g.,hyaluronic acid is an aqueous base); hydrating formulations (e.g.,calendula, vitamins A, D, or E, or any combination, in a mineral oilbase); antioxidant formulations/free radical; scavengers (e.g., vitaminsA, E, and K in a mineral oil base). Other examples of product categoriesthat may be employed alone or in combination with other compoundsinclude, antiseptics, astringents, cleansers, pore decongestants, balms,botanicals, collagen stimulators, herbs, microemulsifiers, oxygendelivery vehicles, proteins, serums, skin firming agents, toners, andtopical anesthetics. Individually named products as may be used (withassociated benefit indicated parenthetically) include: aloe vera(calming); alpha hydroxy acids (peel); alphalipoic acid (antioxidant);benzoil and other peroxides (acne); ceramide (hydrator); copper(toning); copper peptide (toning); CoQ-10 (coenzyme Q-10) and otherenzymes (toning); cortisone (calming); glycolic acids (peel); hyaluronicacid (collagen stimulation); hydrolipids (hydrator); lactic acids(peel); magnesium ascorbic phosphate (free radical scavenger, collagenstimulator, bleaching); niacin (vascular dilation); phospholipids(moisturization); potassium (toning, psoriasis), and salicylic acids(acne). The above ingredients are taught for use in conjunction with USPGPUB 20070088371 to Karasiuk, published Apr. 19, 2007, entitled“Microdermabrasion System and Method of Use.”

As further secondary treatment agents, the tyrosinase inhibitor peptidesand formulation of the present invention can also be optionally mixedwith each other and with other skin whitening agents for purposes oftreatment, i.e., skin lightening or whitening. For example, the skinwhitening products which can be combined include but are not limited tocysteine, 4-thioresorcin, 3-aminotyrosine,5-hydroxy-2-hydroxymethyl-γ-pyridone, fomesjaponicus and ganodermaextracts, kojic acid, glabridin, licorice extract, glycyrrhizinic acid,catharanthus roseus extract, proteoglycans, proteinase inhibitors,oligopeptides, betaines, and methyl 4-benzyloxy-2-hydroxybenzoate,4-benzyloxy-2-hydroxybenzoic acid, etc. The present peptides may also becombined, or combined with other tyrosinase inhibitors, such asisoliquiritigenin chalcone (ILC) or 4,4′-dihydroxybiphenyl (44′-BP)(See, Kim et al. “4,4′-Dihydroxybiphenyl as a new potent tyrosinaseinhibitor,” Biol Pharm Bull. 2005 February;28(2):323-7.)

Dosage

The term “therapeutically effective amount” is intended to mean theamount of drug sufficient to produce a tyrosinase inhibitory effectapplied to a melanocyte, resulting in reduction or elimination of theproduction of melanin. These amounts are known in the art or may bedetermined by methods known in the art, and typically range from about0.1 to 20,000 mg per human adult and preferably about 10 to 10,000 mgand most preferably range from about 20 to 5,000 mg of the inhibitoryagent per application, depending upon the formulation chosen, andwhether the tissue, such as the skin or mucous membrane is the site ofaction. The only upper limit on the amount of anesthetic in thecomposition is that the preparation is substantially free of crystals ofinhibitory agent and the amount of solvent used is not sufficient toundesirably affect the properties of the finite composition allowing ittoo adhere to the desired site of application. Thus, the singleingredient inhibitory peptide contains a therapeutically effectiveamount of agent within the foregoing range. The concentration of peptidehas been found experimentally to be suitable when extrapolated from theIC 50. In general, it is suggested that concentrations above two timesIC 50 would be appropriate for prescription use; below about two timesIC 50 would be suitable for over the counter use. However, one could goup to 100 times IC 50, to allow for lack of skin uptake or other losses.At twice IC 50, 95% tyrosinase inhibition should be achieved. Thefollowing table is exemplary:

Peptide concentration mM grams in 1 oz cream PLG-OH 5.46 0.043 SFpeptide 16 mM 0.379 RA peptide 246 uM 0.0062

The above formulations were prepared essentially as follows:

ACCEPTABLE PREFERRED INGREDIENT NAME RANGE RANGE 1. WATER 1.00-90.00% 30.00-70.00%  2. ALOE BARBADENSIS 1.00-90.00%  5.00-60.00%  LEAF JUICE3. CAPRYLIC/CAPRIC 1.00-15.00%  5.00-10.00%  TRIGLYCERIDE 4. PENTYLENEGLYCOL 0.50-10.00%  1.00-5.00% 5. DIGLYCERIN 0.50-20.00%  1.00-10.00% 6. BIS-ETHOXYDIGLYCOL 0.50-3.00% 1.00-2.00% CYCLOHEXANE1,4-DICARBOXYLATE 7. DIMETHICONE 0.50-10.00%  1.00-5.00% 8. ETHYLASCORBATE 0.10-10.00%  1.00-5.00% 9. SODIUM HYALURONATE 0.50-90.00% 5.00-20.00%  10. SODIUM PCA 0.50-20.00%  1.00-5.00% 11. CETEARYL ALCOHOL0.50-5.00% 1.00-3.00% 12. DICETYL PHOSPHATE 0.50-5.00% 0.50-3.00% 13.CETETH-10 PHOSPHATE 0.50-5.00% 0.50-3.00% 14. GLYCYRRHIZA GLABRA0.01-5.00% 0.10-2.00% (LICORICE) ROOT EXTRACT 15. SQUALANE 0.50-10.00% 1.00-5.00% 16. SCLEROTIUM GUM 0.20-4.00% 0.50-2.00% 17. DECAPEPTIDE-1218. BUTYLENE GLYCOL 1.00-30.00%  3.00-10.00%  19. PANTHENOL 0.10-5.00%0.50-2.00% 20. ALLANTOIN 0.01-1.00% 0.10-0.50% 21. TETRASODIUM EDTA0.05-2.00% 0.10-0.50% 22. CHLORPHENESIN 0.10-1.00% 0.10-0.50% 23.CAPRYLYL GLYCOL 0.10-2.00% 0.50-1.00% 24. PHENOXYETHANOL 0.30-2.00%0.50-1.00%

The concentration as well as the quantity of inhibitory peptide per unitarea, namely per square or cubic centimeter can be varied independentlyin order to achieve the desired effect. Higher concentrations ofinhibitory peptide base contained in a dosage form of decreasedthickness will result in an application of short duration. Highconcentrations of the inhibitory peptide base contained in a dosage formof increased thickness (higher mg of inhibitory peptide per square orcubic centimeter) will result in potent inhibition with fast onset andlong duration. Low concentrations of the inhibitory peptide base in adosage form of decreased thickness will result in mild inhibition withlonger onset and short duration. Low concentrations of the inhibitorypeptide contained in a dosage form of increased thickness will have mildinhibition with longer onset and longer duration. As shown in the aboveexplanation, the ability to vary the concentration of inhibitory peptidefrom very low (about 0.1%) to high (40% or higher) of the totalcomposition, when combined with the ability to coat thin (about 0.001inches) or thick (about 0.500 or more inches) enables the practitionerof the invention to vary the dosage of the system as needed forparticular anatomical sites of interest.

As a general rule, in the case of a given tissue, e.g., thesubepithelial layer, the peptide drug selected, the concentration andthickness and the duration of the application is determined based uponthe peptide's ability to penetrate the tissue, for example the basallayer of the epidermis or mucosa, and to be at peak effectiveness withinabout 2 to 30 minutes. The duration of the effect of the inhibitorypeptide on the tissue, for example the epidermis should range betweenabout 2 to 240 minutes, depending on the agent selected, theconcentration of the inhibitory peptide and the thickness ofapplication. Longer or shorter durations can also be selected dependenton need, as will be apparent to one skilled in the art.

Methods of Treatment

The present peptides, formulated and/or modified as described above, maybe used in a variety of treatment modalities. For example, they may beinjected, ingested or applied in conjunction with laser treatment ordermabrasion/microdermabrasion. Dermabrasion is a cosmetic medicalprocedure in which the surface of the skin is removed by abrasion(sanding). It is used to remove sun-damaged skin and to remove or lessenscars and dark spots on the skin. Dermabrasion units are typicallydiamond tipped, although aluminum crystals are also used. One approach,termed “SilkPeel,” combines a diamond tip micro-dermatome with deepdelivery of solutions, which may include a whitener to improve andrevitalize the skin. In a preferred method, the peptide is administeredas part of a solution delivered during microdermabrasion. If thedermabrasion is carried out with a flow of fluids, which surrounds thearea of skin being microabraded, the skin is both pretreated andpost-treated with the vitamins, lotions, etc., as well as, in apreferred method, the present tyrosinase inhibitor peptide(s).Pretreatment can soften the area of skin treatment to be microabraded,thereby rendering exfoliation more complete and easier to accomplish,with less trauma to the skin tissues left behind, while post-treatmenthelps to reduce streaking and redness of the skin tissues left behind.Further details on this method of treatment may be found in U.S. Pat.No. 6,695,853 to Karasiuk issued Feb. 24, 2004, entitled“Microdermabrasion system and method of use.”

The present peptides may also be used in conjunction with lasertreatment. Laser treatments such as the Erbium laser vaporize variousdepths of damaged skin tissue. Erbium lasers are further described inU.S. Pat. No. 3,978,427. The Erbium laser procedure is performed usingtopical anesthetic solutions and healing is usually two to five daysdepending on the depth of laser energy penetration. Based on theabsorption spectrum of melanin, the Q-switched ruby laser (694 nm) andthe Q-switched Nd:Yag laser (1064 nm) are the lasers of choice for thetreatment of hyperpigmented lesions such as lentigines andpostinflammatory hyperpigmentation in combination with the presentpeptides.

The present peptides may be used in conjunction with a variety ofradiation treatments in addition to laser treatment, such asadministration of radiant energy through RF devices, LEDs, orultrasound. The present peptides may also be used microneedle treatment,electroporation or iontophoresis. An appropriate microneedle isdescribed in U.S. Pat. No. 6,256,533, entitled “Apparatus and method forusing an intracutaneous microneedle array,” issued on Jul. 3, 2001 toGarstein et al. Electroporation involves the application of high voltagepulses to the skin which has been suggested to induce the formation oftransient pores. High voltages and short treatment durations(milliseconds) are most frequently employed. Other electrical parametersthat affect delivery include pulse properties such as waveform, rate andnumber, and are further described in a number of publications. Thetechnology has been successfully used to enhance the skin permeabilityof molecules with differing lipophilicity and size (i.e., smallmolecules, proteins, peptides and oligonucleotides). Iontophoresisinvolves the application of a low level electric current either directlyto the skin or indirectly via the dosage form in order to enhancepermeation of a topically applied therapeutic agent. Increased drugpermeation as a result of this methodology can be attributed to eitherone or a combination of the following mechanisms: Electro-repulsion (forcharged solutes), electro-osmosis (for uncharged solutes) andelectro-perturbation (for both charged and uncharged). Severaliontophoretic systems are currently under commercial development.

In addition to or included with the above mentioned disorders for whichthis invention can be of use, are without limitation: frecklesreduction, reduction of yellow mass-tone on Asians skins and inhibitionof skin, dischromia related to the aging process, as well as a reductionin redness linked to venous disorders and a reduction in UV-inducedpigmentation.

As described above, a preferred method of treatment involves thelightening of skin. The present inhibitors may also be used for othertreatments. Tyrosinase is an attractive target antigen forimmunotherapeutic treatment of patients with melanoma because it is morehomogeneously expressed than several other melanocyte differentiationantigens such as MART-1, gp100, or gp75. In two separate investigations,tyrosinase was found to be expressed in 100% of fresh melanoma specimensevaluated by immunohistochemistry or reverse transcription-polymerasechain reaction. These data indicate that tyrosinase may be an excellenttarget for essentially all patients with melanoma (Riley et al., J.Immunother., 2001, 24, 212-220).

Tyrosinase has also been implicated in Vogt-Koyanagi-Harada (VKH)disease. VKH is a bilateral granulomatous panuveitis associated withcentral nervous system, auditory, and integumentary manifestations. Itusually manifests with prodromal similar to asceptic meningitis,followed by posterior uveitis with exudative retinal detachments anddisk hyperemia. T-cell clones established from patients with VKH diseaseand stimulated with tyrosinase family peptides demonstrated apredominantly proinflammatory, T1-type T-cell response. Read et al.demonstrated that a VKH-like syndrome is inducible in rats byimmunization with peptides derived from tyrosinase and other tyrosinasefamily proteins (Read et al., Curr. Opin. Ophthalmol., 2000, 11,437-442).

EXAMPLES Design and In Vitro Testing

Mushroom tyrosinase, L-tyrosine and other chemicals for the enzymaticreaction were obtained from Sigma-Aldrich. Short sequence peptides 1-7were designed based on potential homology with known tyrosinasesubstrates. All synthetic peptides were between 3 and 10 amino acids inlength and were synthesized using tBoc and/or Fmoc solid phasechemistry. Peptides were confirmed to be of research grade (>80% purity)in all cases. It is understood that research grade reagents were usedfor convenience, and it is preferred that the peptides be prepared topharmaceutical grade purity, greater than 90%, preferably greater than99% pure.

Inhibition of tyrosinase by experimental peptides was determined bycalorimetric detection of dopachrome, the reaction product of thesubstrate L-tyrosine. Mushroom tyrosinase, L-tyrosine, and a potassiumphosphate buffer (pH 6.8) were added to 96-well plates containing shortsequence peptides dissolved in 5% DMSO and incubated at 37° C. Theabsorbance at 475 nm was measured using a BIO-TEK plate reader 30 minafter reaction initiation. Each experiment was performed in triplicateon 3 separate occasions. The protocol is described in further detail inPiao et al., “Mushroom Tyrosinase Inhibition Activity of SomeChromones,” Chem. Pharm. Bull. 56(3): 309-311 (2002), and in Pomerants,J. Biol. Chem 238:2351-2357 (1963).

Enzyme kinetics were calculated using the Michaelis-Menton equation byobserving changes in reaction velocity accompanying substrateconcentrations of 0.5, 1, 2, and 4 mM L-tyrosine. Once initial reactionvelocities were obtained, Lineweaver-Burke plots were created tocalculate Km, Vmax, and to determine the mode of enzymatic inhibition.

IC50 Results

Seven synthetic peptides ranged in molecular weight from 321 too 1,556are presented below. Of the 7 peptides screened, 5 were found to possessvarying inhibitory effects while 2 maintained no activity againsttyrosinase. The IC50 values for these peptides ranged from ˜40 μM to 8mM. Results are given in the Table below:

Molecular weight daltons agent IC50 284.4 PLG-OH 2.73 mM 470.6 VLLK noeffect 838 SFLLRN 8 mM 1200-1400 Poly-L-lactic acid no effect 892.9RADSRADC 123 uM 1200-2400 Poly-L lactic acid no effect

In conclusion, we designed synthetic short sequence peptides based ontheir similarity to observed properties of tyrosinase substrates.Surprisingly, we found peptides that possess varying degrees ofinhibition against tryosinase.

S F L L R N R A D S R A D C P L G

One aspect of the present findings relates to knowledge of possiblepeptide modifications, including the guidance given above. In comparingthe three peptides as shown above, it can be seen that at least onecharged residue internal to the sequence is found in each embodiment.This shows the desirability in the above motif of having positivelycharged residues next to each other, as shown by the underlined D and Rresidues. One may alter sequences based on charge according to knownprinciples, such as the following table:

Group pKA Acids Carboxy-terminus 3.1 Aspartate 4.4 Glutamate 4.4Cysteine 8.5 Tyrosine 10.0 Bases Amino-terminus 8.0 Lysine 10.0 Arginine12.0 Histidine 6.5

Again, without wishing to be bound by any one theory, it is believedthat, for purposes of modification, it appears that PLG is acting incompetition with L-dopa not L-tyrosine. SFLLRN requires thephenylalanine (F) for activity as a L-tyrosine analog. RADSRADC appearsto utilize the terminal cysteine to affect the tryosinase enzyme.

In addition, the presence of one or more amino acids of Tyr or similarresidues (e.g., Phe or Trp) is desirable. These observations providefurther guidance in designing sequences similar to those exemplified.Also, peptides may be designed to form self-assembled secondarystructures, whereby multiple tyrosinase molecules may be bound by asingle complex. This favors sequences having multiple tyrosine residues.

IC50 values were measured to be as low as 40 μM, a value in line withsome of the efficacious tyrosinase inhibitors currently available forcommercial use. The mode of inhibition was determined to be competitivein all cases. Discovery of short sequence peptides with activity againstspecific enzymes represents an important strategy for the development offuture pharmaceutical therapies. Short sequence peptides offer severaladvantages over traditional pharmacological drugs and growth factorsincluding increased skin penetration, amenability to liposomalencapsulation or lipid conjugation, and reduced toxicity. To ourknowledge, this is the first report to demonstrate a significantinhibitory effect of a short sequence peptide against tyrosinase.

Stability Testing

Peptides: Five peptides, including the present >1 SEQ ID NO: 1SFLLRN, >2 SEQ ID NO: 2 RADSRADC, and PLG-OH, were ordered from acommercial supplier, NeoMPS. A sixth peptide, VLLK, was used as innerstandard for quantitative evaluation of other peptide stability.

HPLC/MS: A 1% solution was prepared for each peptide and sealed in atube, which was kept in ambient environment. High Performance LiquidChromatography (HPLC) and Mass Spectroscopy (MS) were testedperiodically at an approximate time range of 30 days. A fresh 1% VLLKsolution used as inner standard was prepared on the same day of eachtest. The amount of each peptide was determined by its peak arearelative to that of VLLK.

Four peptides, including SFLLRN did not show degradation for 4 monthseither from HPLC or MS. However, RADSRADC appears to start degradationduring the first month as indicated by the increase of the peak area ofsmaller species in MS, while after two months, no species of RADSRADCcould be observed from HPLC, indicating that it is completely degraded.It is unclear yet how RADSRADC degraded; the most possible reason mightbe related with cysteine. The mechanism of degradation needs furtherinvestigation, although it suggests that replacement of cysteine withanother amino acid may prove beneficial.

Cellular Assay

Melanogenesis suppressive activity in pigment cell is assayed inaccordance with the method described in Cancer Research, Vol. 42, pp.1994-2002 (1982) with a slight modification. 4×10⁴B-16 cells, a mousemelanoma strain, are suspended in 10 ml Eagle's MEM containing 10 v/v %fetal calf serum, transferred to 25 cm² Roux's flask, and cultured at37° C. in the presence of 5 v/v % CO₂. The culture is continued for 5days while refreshing the culture medium with fresh one additionallycontaining a test specimen on the starting and third days. After washingin phosphate buffer (pH 7.2) containing 0.8 w/v % saline, the cells aredetached with a solution containing trypsin and EDTA, and recovered byfiltration. The cells on the filter paper are then dried, and determinedfor the strength of reflected light at 500 nm using densitometry.

Other assays may be used in testing the present peptides. One mayfurther screen for inhibitors of melanogenesis using assays fortyrosinase activity as described in US PGPUB 2004/0175767 by Orlow, etal., published Sep. 9, 2004, entitled “Methods and compositions thataffect melanogenesis.” As described there, one may cause tyrosinase tobe secreted into a cellular medium for testing. Wild-type melanogeniccells grown in vitro culture will synthesize melanin inside ofmelanosomes as they do in vivo. In these cultured cells, tyrosinase isfound predominantly in the melanosomal membrane, although sometyrosinase is also secreted. The tyrosinase that is found in themelanosomal membrane is held in place by a C-terminal transmembranedomain and has its active site disposed toward the melanosomal lumen. Bycontrast, in melanogenic cells inhibited for melanogenesis througheither a mutation in P protein or a compound that inhibits P proteinfunction, tyrosinase will be mislocalized. A significantly greaterfraction of the cells' tyrosinase is secreted from the cells into thegrowth or incubation medium. Additionally, the secreted tyrosinasepolypeptide will be shorter than that found in wild-type cells becauseit lacks its C-terminal membrane anchor. The secreted tyrosinase,however, is enzymatically active in the growth or incubation mediumwhere it can synthesize melanin from extracellular tyrosine.Consequently, tyrosine-containing growth or incubation media frommelanogenic cells that have been inhibited for melanogenesis will turndark. The higher the concentration of tyrosine in the medium, the darkerthe medium becomes, and the higher the concentration of tyrosinase inthe medium, the faster the medium darkens. Because melanogenic cellsthat are not inhibited for melanogenesis secrete significantly lesstyrosinase, the tyrosine-containing growth or incubation media in whichthey are cultured will not become as dark.

In Vivo Assay

The peptides that exhibit significant tyrosinase inhibition in vitro maybe further tested for their skin-whitening activity in vivo.

In this assay, healthy male and female volunteers (20-50 year old)receive about 0.6 J ultraviolet irradiation at two different spots intheir brachial area, 2.25 cm² each, once every day for 3 days, and aninhibitory peptide is applied on either irradiated spot 3 times everyday over 24 days. Thereafter, the irradiated spot with theskin-whitening agent is compared with control to estimate the degree ofmelanogenic suppression, i.e., skin-whitening effect.

The skin-whitening agent for such an assay may be prepared by mixing 10parts by weight of ethanol and 0.18 parts by weight of methylp-hydroxybenzoate together with either 0 (control), 4, 10, 16 or 40parts by weight of 50 w/w % of either active peptide or a derivativeadjusting the mixture to pH 5.5 with 10 w/w aqueous citric acidsolution, and pouring refined water to the mixture to give a totalamount of 100 parts by weight.

The concentration of peptide in the skin-whitening agent is therefore 0w/w % (control), 2 w/w %, 5 w/w %, 8 w/w % or 20 w/w %.

The skin-whitening agent is applied by first soaking it in gauze, thenattaching the gauze over an irradiated spot in accordance with theocclusive dressing technique.

Skin-whitening effect is determined by comparing the treated spot withcontrol for melanogenic suppression, i.e., skin-whitening effect;grading the skin-whitening effect into either “superior,” “not changed”or “inferior;” and numerating the volunteers answering “superior” (20volunteers in each group).

Clinical Study

Patients with recalcitrant melasma (those that failed over the counterproducts, or HQ 4% for six months, or Tri-luma® Cream for six months)were randomized and blinded to two treatment groups (vehicle andpeptide). Patients applied each product twice per day followed by asunscreen with a sun protection factor of 30 Qam. Patients were thenasked to rate the percentage improvement in their facial pigmentationusing a nominal scale of 0-100, with 0 corresponding to no change and100 corresponding to complete resolution. The results are shown in FIG.3, in which the PLG-OH peptide is shown to be rated as 40-50%improvement, where vehicle caused no improvement. The peptide wasformulated in liposomes at 1%, and the control was the liposomes alone.During this 4-month exposure, patients reported no side effects (eg.erythema, scaling, contact dermatitis reaction, comedone formation) fromthe application of either the vehicle or peptide formulation.

Evaluation of Peptides as Inhibitors of Melanin Synthesis

Peptides SF, VL, and PLG-OH were tested, along with controls of kojicacid, a known tyrosinase inhibitor, and licorice extracts, a knowntyrosinase inhibitor (See J. Agric. Food Chem., 51 (5), 1201-1207,2003).

Tyrosinase inhibition was measured using mushroom tyrosinase (SigmaAldrich cat # T3824) and L-tyrosine (Sigma Aldrich cat # T8566) in anassay buffer consisting of phosphate buffer (1.0M, pH 6.8) and 5%, andabsolute water. The assay buffer, with test sample concentrations andtyrosinase, was incubated in the dark at room temperature for one hourand absorption was read.

The B-16 pigmentation assay used B16-F10 mouse melanoma cells (ATCCCRL-6475) grown in DMEM, DMEM without phenol red, FBS, antibiotics,L-glutamine and other standard components. Cells were counted in aPackard Spectracount (microplate spectrophotometer) filtered at 540 nmand 570 nm. The cells were cultured with growth medium and incubated for24 hrs. at 37° C. and 10% CO₂. Cells were treated with 200 μl of growthmedium containing test active ingredient and incubated at 37° C. and 10%CO₂. After 7 days, the cells were read at 540 nm using the Packardmicroplate reader. Cell viability was assessed using the MTT assay(Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J Immunol Methods.1983 Dec. 16;65(1-2):55-63), which is used to determine the number ofliving cells in a sample.

The results are tabulated as follows:

% Depig- Concentration % Tyrosinase mentation Active tested inhibition(B-16 Assay) Kojic Acid 0.25% 94% ND Licorice Extracts 0.01% 97% NDLicorice Extracts 0.001%  ND 76% SF peptide 8 mM ND (insoluble  0% inbuffer) RA peptide 150 uM 99% 14% RA peptide 300 uM ND 19% RA peptide450 uM ND 23% PLG peptide 3 mM  6% cyto-toxic

As assessed by in vitro Mushroom Tyrosinase activity these data suggestthat RA peptide is an effective inhibitor of Mushroom Tyrosinase whentested at a concentration of 150 uM. This concentration was notsufficient to inhibit melanin synthesis in B 16 cell culture to theextent expected from the previously observed inhibition of Tyrosinaseactivity. However, de-pigmentation increased, albeit moderately, when RApeptide was administered at higher concentrations. Viability was notsignificantly affected at these concentrations. Neither SF peptide norPLG was considered effective in these assays. However, PLG may derive inpart its depigmenting effects from killing melanocytes, which is thesame mechanism of action of HQ. HQ causes 100% cell death at 100 uM, aconcentration 7 fold less than its IC 50 for tyrosinase inhibition.

Detailed Study of RA Peptide Materials

Mushroom tyrosinase, L-tyrosine, hydroquinone and L-DOPA were purchasedfrom Sigma Aldrich. RA peptide (amino acid sequence RADSRADC,purity>82%) was synthesized by NeoMPS, Inc (San Diego, Calif., USA).Melanocytes (passage 3, primary) were obtained as a gift from Dr. ToddRidky in the department of dermatology, Stanford University.

Enzymatic Assay of Mushroom Tyrosinase

Tyrosinase inhibition activity was determined in vitro using L-tyrosineas substrate with a modified method from Piao et al. (Piao L Z, Park HR, Park Y K, Lee S K, Park J H, Park M K. 2002. Mushroom TyrosinaseInhibition Activity of Some Chromones. Chem Pharm Bull 50(3): 309-311.).The concentration of enzyme, substrate and inhibitor was denoted as [E],[S] and [I], respectively. 80 microliters of 0.067 M potassium phosphatebuffer (pH 6.8), 40 microliters of L-tyrosine in 0.067 M potassiumphosphate buffer (pH 6.8), 40 microliters of inhibitor in 5% DMSOsolution, and 40 microliters of mushroom tyrosinase solution were addedto a 96-well microplate, to make the final concentration of each reagentto be: 0.2 mg/ml [S], and 96 units/ml [E], with varying [I]. 5% DMSOsolution instead of an inhibitor solution was added to a blank solutionand adjusted to the total volume of 200 microliters as control. Theassay mixture was incubated at 37° C. The amount of dopachrome producedin the reaction mixture was measured at 475 nm in a microplate reader atdifferent time periods. The percentage of inhibition of tyrosinaseactivity was calculated as follows:

Inhibition (%)=[(A−B)−(C−D)]/(A−B)×100

A: absorbance of blank solution after incubation

B: absorbance of blank solution before incubation

C: absorbance of sample solution after incubation

D: absorbance of sample solution before incubation

Melanocyte Culture and Treatment

Primary human melanocytes were kindly obtained from Dr. Todd Ridky inthe department of dermatology, Stanford University. Cells were culturedin Medium 254 (Cascade Biologics) supplemented with Human MelanocyteGrowth Supplement (HMGS, Cascade Biologics). They were grown in ahumidified atmosphere with 5% CO₂ at 37° C. Cell plating densities werearranged that those cells were in log phase of growth for the durationof incubation with test samples. Subcultures of cells were plated at adensity of 4×10⁴ cells/cm². Approximately 24 h later, fresh medium andtest samples were added. Cells were harvested 7 d after test samplesaddition, replaced with fresh medium and test samples every two days.

Melanocyte Assay for Cell Viability

Cell viability was determined using WST-1 Cell Proliferation Kit(Roche). Cells were plated at 1×10⁵/well (24-well plates). Twenty-fourhours after plating, test samples were added and cultures were incubatedfor an additional 7 d. At the end of the treatment period, 50 μl WST-1was added to each well. The plates were placed at 37° C. for 4 h in thedark and the absorbance at 450 nm was read using microplate. Usually, 3replicate wells were measured for each group to be tested. Wellscontaining medium but no cells served as controls. Cell viability wascalculated according to the equation:

cell viability=absorbance (sample tested)/absorbance (medium only)×100%.

Assay of Cellular Tyrosinase Activity

Cellular tyrosinase activity using L-DOPA as the substrate was assayedby the method of Cheng et al. (Cheng K T, Hsu F L, Chen S H, Hsieh P K,Huang H S, Lee C K, Lee M H. 2007. New Constituent from Podocarpusmacrophyllus var. macrophyllus Shows Anti-tyrosinase Effect andRegulates Tyrosinase-Related Proteins and mRNA in Human EpidermalMelanocytes. Chem Pharm Bull 55(5): 757-761.). Human melanocytes werecultured in 6-well plates. After treatment with individual test samplesfor 7 d, the cells were washed with phosphate-buffered saline (PBS) andlysed with phosphate buffer, pH 6.8, containing 1% Triton X-100. Aftersonication, lysates were clarified by centrifugation at 10000 g for 10min. After determination of protein content with a Bio-Rad protein assaykit, lysates were added to 96 well culture plate containing equalamounts of protein (40 μg), and adjusted with lysis buffer to reach 150μl in each well. 75 μl of 10 mM L-DOPA dissolved in lysis buffer wasadded to each well. The culture plate was incubated at 37° C. for 30min, and then read with the spectrophotometer at 475 nm.

Tyrosinase inhibitory activity was calculated with the followingformula: tyrosinase inhibition (%)=[1−(O.D.475 of sample/O.D.475 ofcontrol)]×100%.

Melanin Content Measurement

Human melanocytes were cultured in 6-well plates and treated withindividual test samples for 7 d. After washes in PBS, cells weredetached by short incubation in trypsin/EDTA (0.25%/0.1% in PBS). Analiquot was used for cell count. The remaining cells were sonicated andincubated overnight in 500 μl 1 M NaOH at 37° C., avoid light. Melaninconcentrations were calculated by comparison of the OD at 475 nm ofunknown samples with a standard curve obtained with synthetic melanin.

Effects of RA Peptide on Cell Viability

The concentrations of HQ tested on melanocytes were 1, 10, 100, and 1000μM, while those of the two peptides were 1, 10, and 100 μM. Melanocyteswere found dead 24 hours after the addition of HQ at either 100 or 1000μM, confirming the cytotoxicity of HQ. Cells were found living with HQtreatment of 10 μM and below. No toxicity was observed with RA peptideat concentrations of up to 100 μM after treatment for 7 days.

To exclude the possibility that the inhibitory effects of RA peptide onmelanogenesis might be caused by the inhibition of cell growth, wecompared the number of cells grown in the presence and absence of testsamples. We did not observe any inhibitory effects of RA peptide onmelanocyte proliferation rates. The proliferation rates after treatmentby different compounds is shown in FIG. 4. Hydroquinone also did notshow inhibitory effects up to 10 μM, although higher concentrationscould not be tested due to 100% cell toxicity.

Effects of RA Peptide on Melanin Synthesis

To provide more direct evidence that RA peptide inhibits melanogenesis,its effects on melanin production in melanocytes was studied. Thecontents of melanin in melanocytes treated with no addition, RA peptideor HQ for 7 days were determined with a spectrophotometer and comparedwith a melanin standard curve. The decrease in melanin content is shownin FIG. 5, showing melanin content of cells as a graph. Treatment with10 μM HQ (non-toxic dose) slightly reduced melanin content. On the otherhand, a similar concentration of peptide significantly reduced melanincontent.

Effects of RA Peptide on Cellular Tyrosinase Activity

We also examined the inhibitory action of RA peptide on cellulartyrosinase activity of melanocytes, using L-DOPA as substrate. Aftercentrifugation and protein calculation, certain volume of supernatantfor each sample was transferred to 96 well culture plate by compensatinga certain protein content in each well, and adjusted the volume of eachwell to 150 μl with 0.067M of phosphate buffer (pH 6.8). 75 μl of 10 mML-DOPA dissolved in phosphate buffer (pH 6.8) was added to each well.The culture plate was incubated at 37° C. for 30 min, and then read withthe spectrophotometer at 475 nm. The results are shown in FIG. 6.Treatment of the melanocytes with 10 μM hydroquinone reduced cellulartyrosinase activity by 28.8%; Treatment with RA peptide led to adose-dependent reduction of cellular tyrosinase activity. At 1 μM, RApeptide reduced enzyme activity by 1.2%; at 10 μM, RA peptide decreasedby 15.6%. Finally, at 100 μM, RA peptide decreased enzyme activity by21.1%.

In summary, the results are presented in FIGS. 4-6 show that the RApeptide did not adversely affect the proliferation of cells; that it hadsignificantly improved reduction of melanin content compared to HQ; andthat it inhibited tyrosinase activity.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention.Any patents or publications mentioned in this specification areindicative of levels of those skilled in the art to which the patent orpublication pertains as of its date and are intended to convey detailsof the invention which may not be explicitly set out but which would beunderstood by workers in the field. Such patents or publications arehereby incorporated by reference to the same extent as if each wasspecifically and individually incorporated by reference, as needed forthe purpose of describing and enabling the method or material referredto.

1. A purified peptide having an IC 50 of tyrosinase of less than about10 mM and selected from the group consisting of: PLG-OH and a sequenceessentially identical to the sequence RADSRADC.
 2. The peptide of claim1 having an IC 50 of less than about 5 mM.
 3. The RA peptide of claim 1wherein one or two residue which are not R or F are substituted with Ror F.
 4. The RA peptide of claim 1 wherein between one and threeresidues are substituted with V, A, L, M or I.
 5. The RA peptide ofclaim 4 wherein a residue which is not R or F is substituted with R orF, there being a total of two to three substitutions.
 6. The RA peptideof claim 1 wherein between one and three amino acids as listed in column1 below is substituted with an amino acid listed in column 2 of thefollowing table, there being a total of one to three substitutions:Column 1 Column 2 R K or L S T, A, A, L, M, I D N, V, A, L, M, I C V, A,L, M, I A G, V, A, L, M, I


7. The peptide of claim 6 where R is not substituted.
 8. The peptidesequence of claim 1 where C has been substituted with V, A, L, M or I.9. The peptide of claim 1 comprising D-amino acids.
 10. The peptide ofclaim 1 linked to a modulating group.
 11. The peptide of claim 10 wherethe modulating group is a fatty acid.
 12. The peptide of claim 11wherein the peptide sequence is RADSRADC or PLG-OH.
 13. The peptide ofclaim 12 where P and L of PLG-OH are D amino acids.
 14. A topical oralor injectable formulation comprising an effective amount of a tyrosinaseinhibitor selected from the group consisting of RADSRADC, PLG-OH, and acompound having no more than 2 amino acid substitutions to RADSRADCaccording to replacement of an amino acid from column 1 with an aminoacid from column 2: Column 1 Column 2 R K or L S T, A, A, L, M, I D N,V, A, L, M, I C V, A, L, M, I A G, V, A, L, M, I


15. The formulation of claim 14 wherein the peptide is RADSRADC.
 16. Theformulation of claim 14 wherein the peptide is PLG-OH.
 17. Theformulation of claim 14 wherein the peptide is at a concentration lessthan about two times the IC
 50. 18. The formulation of claim 14 whereinthe peptide is at a concentration more than about two times the IC 50.19. The formulation of claim 14 which is substantially free of HQ. 20.The formulation of claim 14 further comprising a secondary treatmentagent.
 21. The formulation of claim 20 wherein the secondary treatmentagent is a peptide such that two peptides having two differentstructures within claim 1 are present.
 22. The formulation of claim 14in liquid form.
 23. The formulation of claim 14 further comprising amaterial selected from: hydrating formulations, antioxidantformulations, and free radical scavengers.
 24. The formulation of claim14 wherein the tyrosinase inhibitor consists essentially of RADSRADC.25. The formulation of claim 14 wherein the tyrosinase inhibitorconsists essentially of PLG-OH.
 26. The formulation of claim 14 whereinthe peptide is contained in liposomes.
 27. A dermatologically acceptabletopical formulation comprising an effective amount of a skin whiteningagent and tyrosinase inhibitor selected from the group consisting ofSFLLRN, RADSRADC, PLG-OH, and a compound having no more than 2 aminoacid substitutions to SFLRNN or RADSRADC according to replacement of anamino acid from column 1 with an amino acid from column 2: Column 1Column 2 R K or L S T, A, A, L, M, I D N, V, A, L, M, I C V, A, L, M, IA G, V, A, L, M, I L I, V, A, M, I N D F R, W, Y


28. A method for treatment of skin comprising administering to the skina peptide essentially identical to RADSRADC, SFLLRN or the peptidePLG-OH, wherein said administering of the peptide inhibits tyrosinasesufficiently to lighten skin pigmentation.
 29. The method of claim 28wherein the peptide is RGDSRGDC.
 30. The method of claim 28 wherein thepeptide is SFLLRN.
 31. The method of claim 30 where the SFLLRN containsone or two amino acid substitutions, wherein a residue which is not R orF may be substituted with R or F, and a residue may be substituted withV, A, L, M or I.
 32. The method of claim 29 wherein the administeringcomprises administering a topical preparation.
 33. The method of claim29 wherein the administering comprises administering the peptide andadministering separately with a secondary treatment agent.
 34. Themethod of claim 29 wherein the administering comprises administering inconjunction with a microdermabrasion process.
 35. The method of claim 29wherein the administering is simultaneous with the microdermabrasionprocess.
 36. The method of claim 29 wherein the administering is inconjunction with a radiation process.
 37. The method of claim 29 whereinthe administering is in conjunction with a physical treatment carriedout by an abrading device a microneedle, an electroporation device, oran iontophoretic device.
 38. A kit for carrying out a skin whiteningprocedure, comprising a purified peptide having an IC 50 of tyrosinaseof less than about 10 mM and selected from the group consisting of:PLG-OH and a sequence essentially identical to the sequence of RApeptide; a dermatologically acceptable carrier; a secondary treatmentproduct; and directions for use.
 39. The kit of claim 38 in which thecarrier and the peptide are precombined.